Pharmaceutical compositions and methods employing camptothecins

ABSTRACT

A method for synthesizing camptothecin and camptothecin analogs using a novel hydroxyl-containing tricyclic intermediate and the camptothecin analogs produced by the process. The camptothecin analogs are effective inhibitors of topoisomerase I and show antileukemic and anti-tumor activity.

This is a continuation of application Ser. No. 07/250,094, filed on Sep.28, 1988, now U.S. Pat. No. 4,981,968 which is a continuation-in-part ofSer. No. 07/032,449, filed on Mar. 31, 1987, now U.S. Pat. No.4,894,456.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to camptothecin and analogs thereof which showlife prologation effects in various leukemia systems such as P-388 andL-1210; inhibition of animal tumors such as B-16 melanoma and are potentinhibitors of topoisomerases I and II. It also relates to a method ofsynthesizing the same by means of a novel hydroxyl-containing tricyclicintermediate.

2. Discussion of the Background

Camptothecin is a pentacyclic alkaloid initially isolated from the woodand bark of Camptotheca acuminata by Wall et al (M.E. Wall, M.C. Wani,C.E. Cook, K.H. Palmer, A.T. McPhail, and G.A. Sim, J. Am. Chem. Soc.,94, 388 (1966).

Camptothecin is highly biologically active and displays stronginhibitory activity toward the biosynthesis of nucleic acids.Additionally, camptothecin exhibits potent anti-tumor activity againstexperimentally transplanted carcinoma such as leukemia L-1210 in mice orWalker 256 tumor in rats.

Several methods for the synthesis of camptothecin and camptothecinanalogs are known. These synthetic methods include (i) methods in whichnaturally occurring camptothecin is synthetically modified to produce anumber of analogs and (ii) totally synthetic methods.

U.S. Pat. Nos. 4,604,463; 4,545,880; and 4,473,692 as well as EuropeanPatent Application 0074256 are examples of the former type of syntheticstrategy. Additional examples of this strategy can be found in JapanesePatents 84/46,284; 84/51,287; and 82/116,015. These methods requirednaturally occurring camptothecin which is difficult to isolate and hencethese methods are not suitable for the production of large quantities ofcamptothecin or analogs.

Examples of a variety of totally synthetic routes to camptothecin andcamptothecin analogs can be found in the following references: Sci. Sin.(Engl. Ed), 21(1), 87-98 (1978); Fitoterpapia, 45(3), 87-101 (1974);Yakugaku Zashi, 92(6), 743-6 (1972); J. Org. Chem., 40(14), 2140-1(1975); Hua Hsueh Hsueh Pao, 39(2), 171-8 (1981); J. Chem. Soc., PerkinTrans 1, (5), 1563-8 (1981); Heterocycles, 14(7), 951-3 (1980); J. Amer.Chem. Soc., 94(10), 3631-2 (1972); J. Chem. Soc. D, (7), 404 (1970) andU.S. Pat. No. 4,031,098.

Wani et al, J. Med. Chem., 23, 554 (1980) discloses a synthesis ofcamptothecin and camptothecin analogs which involves the reaction of atricyclic compound with a suitably substituted orthoaminoaldehyde toyield desoxycamptothecin as shown in Equation 1 below. ##STR1##

Desoxycamptothecin is then treated with oxygen to give camptothecinanalogs. A major disadvantage of this procedure is the insolubility ofdesoxycamptothecin and its analogs, requiring large solvent volumes inthe final step. A poor yield of the oxygenation product results underthese conditions.

There exists a need, therefore, for a high-yield, efficient synthesis ofcamptothecin and camptothecin analogs which does not require priorisolation of naturally occurring camptothecin.

A need also exists for a method of synthesizing camptothecin andcamptothecin analogs which does not suffer from insolubility problems ofintermediate compounds and the resulting low yields.

A further need exists for new camptothecin analogs which can besynthesized in an efficient, high-yield manner and which show goodbiological activity.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a methodof synthesizing camptothecin and camptothecin analogs in high yield in atotally synthetic process.

Another object of the present invention is to provide a process forsynthesizing camptothecin and camptothecin analogs which does not sufferfrom problems associated with the insolubility of intermediatecompounds.

A further object of the invention is to provide a process for thepreparation of camptothecin and camptothecin analogs which can be easilymodified to produce a variety of analog structures.

Still a further object of the present invention is to providecamptothecin analogs which show good antitumor activity and otherdesirable biological activities.

These objects and other objects of the present invention which willbecome apparent from the following specification have been achieved bythe present method for the synthesis of camptothecin and camptothecinanalogs, which includes the steps of:

cyclizing a compound of the formula shown below, wherein X is an organicgroup which is converted to a carbonyl group when treated with an acid,##STR2## to form a lactone having the formula ##STR3## deprotecting saidlactone to form a hydroxyl-containing tricyclic compound having theformula shown below, and ##STR4## reacting said hydroxyl-containingtricyclic compound with a substituted ortho-amino compound of theformula ##STR5## wherein n=1-2 and wherein each R is selected from thegroup consisting of cyano, methylenedioxy, formyl, hydroxy, C₁₋₈ alkoxy,nitro, amino, chloro, bromo, iodo, fluoro, C₁₋₈ alkyl, trifluoromethyl,aminomethyl, azido, amido and hydrazino groups; R² is H₁ or C₁₋₈ alkyl;and R³ is the side-chain of any of the twenty naturally occurring aminoacids.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1, 1A-2, and 1B illustrate the synthesis of thehydroxyl-containing tricyclic compound 11, according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The camptothecin and camptothecin analogs produced by the process of thepresent invention are racemic, and therefore contain both the(R)-20-hydroxy and (S)-20-hydroxy camptothecin compounds. Naturallyoccurring camptothecin belongs to the 20(S) series of compounds.Therefore, the compounds produced by the process of the presentinvention contain a mixture of the natural and non-naturally occurringcompounds.

The camptothecin analogs of the present invention have the basiccamptothecin structural framework shown below in which the A ring issubstituted. ##STR6##

Substituents within the scope of the present invention include hydroxy,nitro, amino, chloro, bromo, iodo, fluoro, C₁₋₈ alkyl, C₁₋₈ alkoxy,trifluoromethyl, aminomethyl, amido, hydrazino, azido, formyl, and cyanogroups as well as groups comprising amino acids and/or peptides bondedto the aromatic ring via the amino-nitrogen atom or via an amide linkagewhich contains the carbonyl group of the amino acid, i.e., an amino acidamido group. Amides prepared from cyclic anhydrides may also beprepared. Preferred alkyl groups include methyl, ethyl, propyl, butyl,isopropyl, isobutyl and sec-butyl groups. Preferred alkoxy groupsinclude methoxy, ethoxy, propoxy and isopropoxy groups.

The preferred amino acid groups are the 20 naturally occurring aminoacids having an (L) configuration. These amiono acids are well known tothose skilled in the art.

Water-soluble camptothecin analogs which can be prepared by the processof the present invention include analogs in which R_(n) is an amidelinkage formed by reacting an amino-camptothecin analog with the freecarboxylic acid group of an amino acid, peptide or carboxylic acidderivative thereof. These amide derivatives may be present as the freeamines, i.e., the α-amino group or as any of the well known acidaddition salts, such as, for example, hydrochloride, gluconate,phosphate or hydrobromide addition salts. Additional water solubleanalogs can be prepared by reacting an amino-camptothecin analog with acyclic carboxylic acid anhydride to give a carboxylic acid amide group.This reaction results in an amide substituent on the camptothecinstructural framework and a free carboxylic acid group which may bepresent as the free acid or as a salt, e.g., alkali metal oralkaline-earth metal salt, or as an organic cation, e.g., ammoniumsalts. Preferred anhydrides are the C₄₋₁₀ saturated and unsaturated acidanhydrides. Alternatively, the above water soluble derivatives can alsobe prepared by employing the corresponding ester-acid halide in place ofthe anhydride. Water soluble urea and urethane analogs can also beprepared by reacting an amino-camptothecin analog with phosgene followedby an appropriate diamine, e.g. n-alkylpiperazine or an appropriatetertiary-amino alcohol, e.g. n-dialkyl-aminoethannol respectively. Thesewater soluble derivatives are particularly important since they may betherapeutically administered in aqueous pharmaceutical compositions.

Additionally, water soluble dialkylamino ether analogs of camptothecincan be prepared by reacting an appropriate tertiary-aminoalkyl halide,e.g. dialklaminoethyl halide with an hydroxy-camptothecin analogfollowed by salt formation as described above.

Additionally, two substituents on the A ring may be joined together toform a bifunctional substituent such as the methylenedioxy group.Methylenedioxy substituents may be bonded to any two consecutivepositions in the A ring, for example, the 9,10; 10,11 or 11,12positions.

Preferred substituents include the hydroxy, amino, cyano,methylenedioxy, 9 or 10-glycinamido, 9-or 10-succinamido, 9-or10-(4-methylpiperazino) carbonylamino, 9-or 10-(N,N-diethylaminoethoxy)carbonylamino, and 9-diethylaminoethoxy substituents. A particularlypreferred substituent is the methylenedioxy group.

Particularly preferred compounds within the scope of the inventioninclude 11-methoxy-20(RS)-camptothecin, 11-hydroxy-20(RS)-camptothecin,10-hydroxy-20(RS)-camptothecin, 9-methoxy-20(RS)-camptothecin,9-hydroxy-20(RS)-camptothecin, 10-nitro-20(RS)-camptothecin,10-amino-20(RS)-camptothecin, 9-nitro-20(RS)-camptothecin,9-amino-20(RS)-camptothecin, 11-nitro-20(RS)-camptothecin,11-amino-20(RS)-camptothecin, 10,11-dihydroxy-20(RS)-camptothecin,10-chloro-20(RS)-camptothecin, 10-methyl-20(RS)-camptothecin,11-formyl-20(RS)-camptothecin and 11-cyano-20(RS)-camptothecin,10,11-methylenedioxy-20(RS)-camptothecin, 9-or10-glycinamido-20(RS)-camptothecin, 9-or10-succinamido-20(RS)-camptothecin, 9- or 10-(4-methylpiperazino)carbonylamino-20(RS)-camptothecin, 9-or 10-(N,N-diethylaminoethoxy)carbonylamino-20(RS)-camptothecin, and9-diethylaminoethoxy-20(RS)-camptothecin.

Also included within the scope of the present invention are compounds inwhich the A ring of the camptothecin structure is modified to contain ahetero atom. The modified structures can have an A ring which contains 5or 6 atoms and the hetro atom may be a nitrogen, sulfur or oxygen atom.These compounds may be represented by the general structure shown belowin which the A ring is an aromatic 5 or 6 membered ring containing thehetero atom X. ##STR7##

Preferred compounds having a modified A ring structure include compoundsin which the A ring is a 6 membered nitrogen-containing aromatic ringand compounds in which the A ring is a 5 membered sulfur-containingaromatic ring. Particularly preferred compounds are10-aza-20(RS)-camptothecin and A-nor-9-thia-20(RS)-camptothecin.

The camptothecin analogs noted above may be synthesized according to themethod of the present invention by reacting a tricyclic compoundcontaining a 20-hydroxyl group with an appropriately substitutedortho-amino aromatic aldehyde or ketone. Camptothecin analogs having analkyl substituent on C₇ are produced when the appropriate ortho-aminoketone is used.

An important step in the method of the present invention is thesynthesis of the hydroxyl-containing tricyclic compound having theformula I shown below and in which R is a hydroxyl group. ##STR8##

A synthetic method previously developed by the present inventors (J.Med. Chem., 23, 554 (1980)) utilized a related by structurally differenttricyclic compound (formula I, R=H). In that method, the tricycliccompound was reacted with a suitable orthoaminoaldehyde under alkalineor acidic conditions to yield a desoxycamptothecin. Thedesoxycamptothecin was then reacted with oxygen to give camptothecinanalogs in which R is OH. A major disadvantage of this procedure is theinsolubility of the desoxycamptothecin and its analogs, requiring largesolvent volumes in the final step and giving poor yields of theoxygenation product.

In contrast, the method of the present invention synthesizes the keytricyclic intermediate (11) according to FIG. 1. The synthesis ofcompounds 1-9 was disclosed in Wani et al, J. Med. Chem., 23, 554(1980). In further contrast to the previous synthesis, the presentmethod introduces the 20-hydroxyl group earlier in the syntheticsequence and then forms the lactone ring to give compound 10. Afterdeprotection of the carbonyl group, the key hydroxyl-containingtricyclic compound 11 is obtained.

The protection of the carbonyl group in compound 3 can be performedusing any appropriate organic protecting group which can be removed orconverted into a carbonyl group upon treatment with acid. The carbonylgroup is thereby "deprotected". These protecting groups are well knownto those familiar with synthetic chemistry, and include acetals, ketals,thioacetals, thioketals, etc. Preferred protecting groups have 2-6carbon atoms. An especially preferred protecting group is --OCH₂ CH₂O--.

As a consequence of prior introduction of the hydroxyl group into thetricyclic compound 11, the desired pentacyclic analogs are produced inone step by reaction with the appropriate ortho-amino carbonylcompounds. Both compound 11 and the corresponding ketonic synthons arevery soluble in organic solvents whereas the pentacyclic product isinsoluble. Hence, the oxygenation step, i.e., the introduction of thehydroxyl group, is conveniently carried out at the tricyclic stagerather than on the insoluble pentacyclic desoxy analogs.

Tricyclic compound 11 is then reacted with a suitably substitutedortho-amino aldehyde or ketone to give a camptothecin analog.Substituted ortho-amino aldehydes and ketones within the scope of thepresent invention include ortho-amino aldehydes and ketones having atleast one additional substituent on the aromatic ring. This substituentmay be at one or more of the positions equivalent to the 9, 10, 11 or 12positions of the A ring of the final camptothecin structure as shownbelow. ##STR9##

Preferred substituted ortho-amino aldehydes and ketones havesubstituents in one or more of the equivalent 9, 10, or 11 positions.

The substituents on the substituted ortho-aminoaldehyde or ketoneinclude hydroxy, nitro, amino, C₁₋₈ alkyl, chloro, bromo, iodo, fluoro,methylenedioxy (--O--CH₂ --O--), C₁₋₈ alkoxy, trifluoromethyl,aminomethyl, amido, hydrazino, azido, formyl, and cyano groups as wellas groups comprising amino acids bonded to the aromatic ring through theamino-nitrogen atom. Preferred examples include the hydroxy, amino,cyano and methylenedioxy substituents. A particularly preferredsubstituent is the methylenedioxy group.

When an ortho-amino ketone is reacted with tricyclic compound 11, acamptothecin analog having an alkyl substituent at C₇ is produced.Preferred ortho-amino ketones are those in which R² is an alkyl grouphaving 1-8 carbon atoms. Especially preferred ortho-amino ketones areortho-aminoacetophenone and ortho-aminopropiophenone.

The ortho-amino aldehydes and ketones may be substituted by a grouphaving the formula

    --NH--CHR.sup.3 --COOH

wherein R³ is a side-chain of one of the twenty naturally occurringamino acids. The amino acid substituent is bonded to the aromatic ringvia the nitrogen atom and may be bonded to any position on the aromaticring equivalent to the 9, 10, 11 or 12 positions of the A ring of thefinal camptothecin structure.

The ortho-amino aldehydes and ketones may be in the free carbonyl formor in a form in which the carbonyl of the aldehyde or ketone isprotected by a standard protecting group. These protecting groups arewell known to those skilled in the art. Ortho-amino aldehydes andketones in the free carbonyl form and in the protected carbonyl form areconsidered within the scope of the present invention and are suitablefor use in the present method.

The reaction in which the hydroxyl group is introduced into thetricyclic intermediate compound, i.e., the cyclizing step, can beeffected by any suitable reaction which will introduce the hydroxylgroup at the appropriate position of compound 9 without causingsignificant side reactions such as degradation of compound 9 itself.

The reaction is preferably conducted in the presence of a basiccatalyst. Suitable basic catalysts include both inorganic and organicbases. Preferred inorganic bases include, for example, sodium andpotassium carbonate and sodium and potassium bicarbonate. Preferredorganic bases include hindered bases such as triethylamine anddiisopropylamine. A particularly preferred basic catalyst is potassiumcarbonate.

The reaction in which the hydroxyl group is introduced can be performedin the presence of any polar or non-polar solvent in which the reactantsare suitably soluble to react. Preferred are polar organic solvents suchas methanol, ethanol, propanol, butanol and dimethylformamide. Ethersolvents, including crown ethers may also be used.

The oxygen of the hydroxyl group is generally derived from molecularoxygen which is bubbled through the reaction solution. Although the useof oxygen is preferred, other sources of oxygen, such as air, may alsobe used. Other oxidizing agents such as hydrogen peroxide, leadtetraacetate and selenium dioxide may also be employed.

This reaction is preferably conducted at room temperature although thespecific reaction temperature will be dependent on the specific reactionconditions and reactants used.

The deprotection of the carbonyl group in compound 10 is accomplished bytreatment with acid. Suitable acids include mineral acids such as HCl,H₂ SO₄, HNO₃, and H₃ PO₄, as well as organic acids such as alkanoicacids having 1-10 carbon atoms, preferably acetic acid, and C₁₋₁₂arylsulfonic acids, especially p-toluenesulfonic acid. The deprotectionof a carbonyl group in this manner is well known to those skilled in theart.

The tricyclic compound 11 is then reacted with a substituted ortho-aminoaldehyde or ketone in the presence of an acid or base catalyst. The basecatalyst is preferably any of the base catalysts noted above incyclizing compound 9 to form compound 10, i.e., for the introduction ofthe hydroxyl group into tricyclic compound 11. The acid catalyst ispreferably a mineral acid such as for example HCl, H₂ SO₄, HNO₃, and H₃PO₄, or organic acids such as C₁₋₈ alkanoic acids and C₁₋₁₂ arylsulfonicacids, especially p-toluenesulfonic acid.

The reaction of compound 11 with an appropriate ortho-amino compound maybe carried out near or in the presence of a polar or non-polar solvent.Preferred polar solvents are the C₁₋₆ alcohols, ethers anddimethylformamide. Preferred non-polar solvents are branched or straightchained alkyl hydrocarbons having 4-10 carbon atoms and aromatichydrocarbons having 6-20 carbon atoms. An especially preferred solventis toluene.

The reaction of the hydroxyl-containing tricyclic compound with theoptionally substituted ortho-amino compound is generally conducted withheating at reflux. Reaction times will vary depending on the particularreactants but are generally in the range from about 10 minutes to 24hours. Preferred reaction times are in the range of 2-10 hours.

The camptothecin analogs of the present invention have excellentbiological activity. As used herein, "biological activity" refers to theability of the camptothecin analogs to inhibit topoisomerase enzymes, inparticular topoisomerase I, and their ability to exert anti-leukemicactivity. Anti-leukemic activity may be determined by the ability of therespective compounds to inhibit L-1210 mouse leukemia cells. Althoughanti-leukemic activity is demonstrated here by the activity of theparticular compounds against L-1210 mouse leukemia cells, other knownanti-leukemic and anti-tumor in vitro and in vivo models may be used aswell to determine anti-leukemic activity.

The mouse anti-leukemic activity of the various ring A oxygenatedcamptothecin analog is shown in Table I. Similar data for nitrogenanalogs and for ring A modified analogs are shown in Tables II and III,respectively. In most cases camptothecin or an analog with well-definedactivity was also assayed at the same time as a positive control, andthe data are shown in the table footnotes. In this manner the relativeantileukemic activity of the various compounds can be compared. Thebiological activity of additional camptothecin analogs is described inJ. Med. Chem., 23, pages 554-560 (1980) incorporated herein byreference.

The ability of camptothecin to inhibit topoisomerase I has been shown.See J. Biol. Chem., 260, 14873-73 (1985) incorporated herein byreference.

                                      TABLE I                                     __________________________________________________________________________    Comparative Activities and Potencies of Ring A Oxygenated                     Camptothecin Analogues in Mouse Leukemia Assays.sup.a,b                       Camptothecin                                                                              max % T/C                                                                            no. cures                                                                          K.sub.E.sup.c at max                                                                active dose                                                                          toxic dose,                              derivative  (dose, mg/kg)                                                                        out of 6                                                                           % T/C range, mg/kg                                                                         mg/kg                                    __________________________________________________________________________    10-OH-20(S).sup.a,d                                                                       297 (3.1)                                                                            0          0.4.sup.e -3.1                                                                       6.25                                     10-OMe-20(S).sup.a,d                                                                      167 (1.6)                                                                            0          0.4.sup.e -1.6                                                                       3.1                                      11-OH-20(RS).sup.b,f                                                                      357 (60.0)                                                                           3    ≧5.68                                                                        7.5.sup.e -60.0.sup.g                                                                >60.0                                    10,11-diOMe-20(RS).sup.b,h                                                                inactive                 >50.0.sup.g                              10,11-OCH.sub.2 O-20(RS).sup.b,i                                                          325 (2.0)                                                                            2    ≧5.97                                                                        2.0.sup.e -4.0                                                                       >8.0                                     10-OCH.sub.2 CO.sub.2 Na-20(S).sup.a,d                                                    inactive                                                          10-Et.sub.2 N(CH.sub.2).sub.2 O-20(S).sup.a,d                                             183 (16.0)                                                                           0          2.0.sup.e -32.0.sup.g                                                                >32.0                                    __________________________________________________________________________     .sup.a Denotes testing in P388 system; treatment schedule Q04DX03; % T/C      survival time of treated/control animals × 100; IP using Klucel         emulsifier.                                                                   .sup.b Denotes testing in L1210 system; treatment schedule Q04DX02; % T/C     = survival time of treated/control animals × 100; IP using Klucel       emulsifier.                                                                   .sup.c Log.sub.10 of initial tumor cell population minus log.sub.10 of        tumor cell population at end of treatment.                                    .sup.d 20(S)Camptothecin and 20(S)camptothecin sodium were used as            reference standards; for 20(S)camptothecin, % T/C (4.0 mg/kg) = 197; for      40(S)camptothecin sodium, % T/C (40.0 mg/kg) = 212.                           .sup.e Lowest dose administered.                                              .sup.f 20(S)camptothecin and 20(S)camptothecin sodium were used as            reference standards: for 20(S)camptothecin, % T/C (8.0 mg/kg) = 164; for      20(S)camptothecin sodium, % T/C (40.0 mg/kg) = 178.                           .sup.g Highest dose administered.                                             .sup.h 20(S)camptothecin sodium was used as a reference standard: % T/C       (25.0 mg/kg) = 206.                                                           .sup.i 20(S)camptothecin was used as a reference standard: % T/C (5 mg/kg     = 166.                                                                   

                                      TABLE II                                    __________________________________________________________________________    Comparison of Activities and Potencies of Ring A Nitrogen Substituted and     Ring A                                                                        Nitrogen/Oxygen Disubstituted Analogues in L-1210 Mouse Leukemia              Assays.sup.a                                                                  Camptothecin                                                                              max % T/C                                                                            no. cures                                                                          K.sub.E.sup.b at max                                                                active dose                                                                          toxic dose,                              derivative  (dose, mg/kg)                                                                        out of 6                                                                           % T/C range, mg/kg                                                                         mg/kg                                    __________________________________________________________________________    10-NO.sub.2 -20(RS).sup.c                                                                 219 (15.5)                                                                           1    ≧5.86                                                                        7.5.sup.d -15.5                                                                      31.0                                     10-NH.sub.2 -20(RS).sup.c                                                                 329 (8.0)                                                                            3    ≧5.86                                                                        4.0.sup.d -16.0                                                                      32.0                                     10-NHAc-20(RS).sup.c                                                                      318 (40.0)                                                                           1    ≧5.86                                                                        5.0.sup.d -40.0.sup.e                                                                >40.0                                    9-NO.sub.2 -20(S).sup.f                                                                   348 (10.0)                                                                           5    ≧5.86                                                                        2.5.sup.d -20.0                                                                      40.0.sup.e                               12-NO.sub.2 -20(S).sup.f                                                                  151 (40.0)                                                                           0    0.34  2.5.sup.d -40.0.sup.e                                                                >40.0                                    9-NH.sub.2 -20(S).sup.f                                                                   348 (2.5)                                                                            4    ≧5.86                                                                        2.5.sup.d -5.0                                                                       10.0                                     12-NH.sub.2 -20(S).sup.f                                                                  inactive                                                          9-NO.sub.2 -10-OMe-20(S).sup.f                                                            160 (40.0)                                                                           0    1.03  2.5.sup.d -40.0.sup.e                                                                >40.0                                    9-NH.sub.2 -10-OMe-20(S).sup.f                                                            186 (40.0)                                                                           0    2.92  2.5.sup.d -40.0.sup.e                                                                >40.0                                    9-NO.sub.2 -10-OH-20(S).sup.f                                                             131 (20.0)                                                                           0    -1.12 2.5.sup.d - 40.0.sup.e                                                               >40.0                                    9-NHAc-10-OH-20(S).sup.f                                                                  220 (40.0)                                                                           0    5.50  2.5.sup.d -40.0.sup.e                                                                >40.0                                    __________________________________________________________________________     .sup.a Treatment schedule Q04DX2; % T/C = survival time of treated/contro     animals × 100; IP using Klucel emulsifier.                              .sup.b Log.sub.10 of initial tumor cell population minus log.sub.10 of        tumor cell population at end of treatment.                                    .sup.c 20(S)Camptothecin (1) and 10hydroxy-20(S)-camptothecin (2) were        used as reference standards: for 1, % T/C (8.0 mg/kg) = 197; for 2, % T/C     (24.0 mg/kg) = 230.                                                           .sup.d Lowest dose administered.                                              .sup.e Highest dose administered.                                             .sup.f Compounds 1 and 2 were used as reference standards: for 1, % T/C       (10.0 mg/kg) = 267; for 2, % T/C (20.0 mg/kg) = 348.                     

                                      TABLE III                                   __________________________________________________________________________    Comparative Activities of Ring A Modified and Homologated                     Camptothecin Analogues in Mouse Leukemia Assays.sup.a,b                       Camptothecin                                                                              max % T/C                                                                            no. cures                                                                          K.sub.E.sup.c at max                                                                active dose                                                                          toxic dose,                              derivative  (dose, mg/kg)                                                                        out of 6                                                                           % T/C range, mg/kg                                                                         mg/kg                                    __________________________________________________________________________    10-aza-20(RS).sup.a,d                                                                     162 (2.5)                                                                            0    1.61  1.25.sup.e -2.5                                                                      5.0                                      12-aza-20(RS).sup.b,f                                                                     175 (32.0)                                                                           0          8.0-32.0                                                                             >32.0                                    A-nor-9-thia-20(RS).sup.a,d                                                               193 (25.0)                                                                           0    -0.94 3.12.sup.e -25.0                                                                     50.0.sup.g                               __________________________________________________________________________     .sup.a Denotes testing in L1210 system; treatment schedule Q04DX2; % T/C      survival time of treated/control animals × 100; IP using Klucel         emulsifier.                                                                   .sup.b Denotes testing in P388 system; treatment schedule Q04DX3; % T/C =     survival time of treated/control animals × 100; IP using Klucel         emulsifier.                                                                   .sup.c Log.sub.10 of initial tumor cell population minus log.sub.10 of        tumor cell population at end of treatment.                                    .sup.d 20(S)Camptothecin sodium was used as a reference standard: % T/C       (40.0 mg/kg) = 215.                                                           .sup.e Lowest dose administered.                                              .sup.f 20(S)Camptothecin was used as a reference standard: % T/C (4.0         mg/kg) = 197.                                                                 .sup.g Highest dose administered.                                        

Pharmaceutical compositions containing the novel camptothecin analogsare also within the scope of the present invention. These pharmaceuticalcompositions may contain any quantity of a camptothecin analog which iseffective to inhibit topoisomerase I in vitro or in vivo or exhibitanti-leukemic activity in vivo. Mammals such as humans are treatablewith the inventive compositions. Typical in vivo doses within the scopeof the invention are from 0.1-60 mg of camptothecin analog per kg ofbody weight. A particularly preferred range is 1-40 mg/kg.

There may also be included as part of the composition pharmaceuticallycompatible binding agents, and/or adjuvant materials. The activematerials can also be mixed with other active materials which do notimpair the desired action and/or supplement the desired action. Theactive materials according to the present invention can be administeredby any route, for example, orally, parenterally, intravenously,intradermally, subcutaneously, or topically, in liquid or solid form.

A preferred mode of administration of the compounds of this invention isoral. Oral compositions will generally include an inert diluent or anedible carrier. They may be enclosed in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, theaforesaid compounds may be incorporated with excipients and used in theform of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like. These preparations should contain atleast 0.1% of active compound but may be varied depending upon theparticular form.

The tablets, pills, capsules, troches and the like may contain thefollowing ingredients: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, corn starch and thelike; a lubricant such as magnesium stearate or Sterotes; a glidant suchas colloidal silicon dioxide; and a sweetening agent such as sucrose orsaccharin or flavoring agent such as peppermint, methyl salicylate, ororange flavoring may be added. When the dosage unit form is a capsule,it may contain, in addition to material of the above type, a liquidcarrier such as a fatty oil. Other dosage unit forms may contain othervarious materials which modify the physical form of the dosage unit, forexample, as coatings. Thus tablets or pills may be coated with sugar,shellac, or other enteric coating agents. A syrup may contain, inaddition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors. Materials used inpreparing these various compositions should be pharmaceutically pure andnon-toxic in the amounts used.

For the purposes of parenteral therapeutic administration, the activeingredient may be incorporated into a solution or suspension.

The solutions or suspensions may also include the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

The dosage values will vary with the specific severity of the diseasecondition to be alleviated. Good results are achieved when the compoundsdescribed herein are administered to a subject requiring such treatmentas an effective oral, parenteral or intravenous dose. It is to beunderstood that for any particular subject, specific dosage regimensshould be adjusted to the individual need and the professional judgmentof the person administering or supervising the administration of theaforesaid compound. It is to be further understood that the dosages setforth herein are exemplary only and they do not limit the scope orpractice of the invention. The dosages may be administered at once, ormay be divided into a number of smaller doses to be administered atvarying intervals of time.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES Synthesis of Tricyclic Compound 116-Cyano-7-methyl-1,5-dioxo-Δ⁶(8) -tetrahydroindolizine (Compound 3)

Ethyl acetopyruvate was prepared from acetone and diethyl oxalate asdescribed in Org. Synthesis, Coll. Vol. 1, 238 (1958). Further reactionwith triethylorthoformate and ammonium chloride in ethanol afforded theknown enol ether 1. See L. Claisen, Chem. Ber., 40, 3903 (1907).

Ethyl(2-ethoxy-4-oxo)-pent-2-enoate(1) (100.01 g, 0.538 mol) was addedgradually to a preheated (45°), mechanically stirred mixture of K₂ CO₃(79.04 g, 0.573 mol) and cyanoacetamide (48.46 g, 0.577 mol) in DMF(960) mL). The mixture was kept at 45° for 18 h, whereupon the thick,red slurry was treated dropwise with freshly distilled methyl acrylate(360 mL, 343 g, 3.99 mol). After 72 hr at 45°, the red suspension wasfiltered, dissolved in 5 liters of water, and acidified to pH 1.5 withconcentrated HCl. Crude bicyclic ester 2 (127.98 g) was collected byfiltration as a pink solid. Without further treatment, 2 was refluxed ina solution concentrated of HCl (800 ml) and glacial HOAc (800 ml) for 2hr. Removal of the solvents in vacuo gave the bicyclic pyridone 3 (39.66g, 39% based on 1).

6-Cyano-1,1-(ethylenedioxy)-7-methyl-5-oxo-Δ⁶(8) -tetrahydroindolizine(Compound 4)

Compound 3 (10.54 g, 0.056 mol) as a stirred solution in CH₂ Cl₂ (500ml) was treated at room temperature under N₂ with ethylene glycol (6.85ml, 7.63 g, 0.123 mol) and Me₃ SiCl (31.30 ml, 26.39 g, 0.247 mol) andleft at ambient temperature (20°) for 65 hr. The solution was filteredto remove some black suspended material before washing with 1M aq NaOHsolution. The organic phase was washed with brine, filtered throughCelite and evaporated to afford ethylene ketal 4 (10.26 g, 79%) as apink solid.

6-Cyano-1,1-(ethylenedioxy)-7-[(ethoxycarbonyl)methyl]-5-oxo-Δ⁶(8)-tetrahydroindolizine (Compound 5)

The ketal 4 (5.0 g, 0.022 mol) was refluxed in a suspension of KH (11.9g, 0.068 mol) in toluene (40 mL) for 10 min. Diethyl carbonate (6.79 g,0.058 mol) and a catalytic amount (0.31 g, 6.7 mmol) of absolute ethanolwere added and refluxing continued for 3 hr. The dark solid was crushedand the resulting suspended salt of 5 was collected by filtration. Thesalt was neutralized by the careful addition of cold aqueous HOAc. Waterwas added and the product extracted into CH₂ Cl₂. Following a wash withbrine and drying (Na₂ SO₄), evaporation of the CH₂ Cl₂ afforded crude 5.Purification by silica gel chromatography (2% MeOH in CHCl₃) andrecrystallization (MeOH) gave pure 5 (4.97 g, 76%).

6-Cyano-1,1-(ethylenedioxy)-7-[1'(ethoxycarbonyl)propyl]-5-oxo-Δ.sup.6(8)-tetrahydroindolizine (Compound 6)

A stirred solution of the ester 5 (4.01 g, 0.0132 mol) in anhydrous DME(70 mL) at -78° C. was treated with potassium tert-butoxide (1.7 g, 15mmol). After 5 min, EtI (8.24 g, 0.053 mol) was added over a 5 minperiod. After stirring for 1.5 hr at -78° C., the mixture was left towarm to room temperature overnight. Water was added and the productextracted into CH₂ Cl₂. After washing with brine and drying (Na₂ SO₄),CH₂ Cl₂ was evaporated to give the ester 6 (4.3 g, 98%).

6-(Acetamidomethyl)-1,1-(ethylenedioxy)-7-[1'-(ethoxycarbonyl)propyl]-5-oxo-Δ⁶(8)-tetrahydroindolizine (Compound 7)

A solution of the ester ketal 6 (2.0 g, 6.0 mmol) in acetic anhydride(30 mL) and HOAc (10 mL) was hydrogenated for 6 hr at 45° C. under 50psi in the presence of Raney nickel (3 g; washed with HOAc). Thecatalyst was removed by filtration and the solvent removed in vacuo togive 7 (2.3 g, 100%) as an oil. Purification by silica gel columnchromatography (2% MeOH in CHCl₃) gave pure 7 as an oil.

6-(Acetoxymethyl)-1,1-(ethylenedioxy)-7-[1'-(ethoxycarbonyl)propyl]-5-oxo-.DELTA.⁶(8)-tetrahydroindolizine (Compound 9)

A cooled solution of amide 7 (2.3 g, 6.0 mmol) in Ac₂ O (30 mL) and HOAc(10 mL) was treated with NaNO₂ (1.8 g, 26 mmol) and the reaction mixturestirred for 2 h at 0° C. Inorganic salts were removed by filtration andthe solvent removed in vacuo at room temperature to afford the N-nitrosointermediate 8 as an oil. Compound 8 was converted directly to the titleacetoxy compound 9 by refluxing overnight in CCl₄. The solution waswashed with water, dried (Na₂ SO₄) and the solvent removed in vacuo togive 9 (2.3 g 100%) as an oil.

1,1'Ethylenedioxy-5-oxo-(5'-ethyl-5'-hydroxy-2'H,5'H,6'H-6-oxopyrano)-[3',4'-f]-Δ⁶,8-tetrahydroindolizine (Compound 10)

Oxygen was bubbled through a mixture of6-(acetoxymethyl)-1,1-(ethylenedioxy)-7-[1'-ethoxycarbonyl)-propyl]-5-oxo-Δ⁶,8-tetrahydroindolizine (Compound 9, 405 mg, 1.07 mmol), anhydrous K₂ CO₃(148 mg 1.07 mmol) and methanol (7.5 mL) for 24 hr. The solution wascooled in an ice bath and made acidic (pH 2-4) by addition of 1N H₂ SO₄.Most of the methanol was removed in vacuo at room temperature, and water(20 mL) was added. The aqueous solution was extracted with CH₂ Cl₂ (3×20mL), dried (NaSO₄) and evaporated to give a solid which was crystallizedfrom CH₂ Cl₂ -hexane to give 280 mg (85%) of 10: mp 179°-181° C.;ν_(max) (CHCl₂) 1740, 1660 cm⁻¹ ; ¹ H-NMR (CDCl₃) δ0.91 (t, 3, J=7 Hz,CH₂ CH₃ ), 1.75 (q, 2, J=7 Hz, CH₂ CH₃), 2.35 (t, 2, J=6.5 Hz, CH₂ α toketal), 4.1 (m, 6, OCH₂ CH₂ O and CH₂ N), 5.30 (m, 2, ArCH₂ O), 6.87 (s,1, pyridone). Anal. Calcd for C₁₅ H₁₇ NO₆ : C, 58.63; H, 5.54; N, 4.56.Found: C, 58.72, H, 5.68; N, 4.57.

5'RS-1,5-Dioxo-(5'-ethyl-5'-hydroxy-2'H,5'H,6'H-6-oxopyrano)-[3',4',f]-.DELTA.⁶,8-tetrahydroindolizine (Compound 11)

A solution of 10 (3.88 g, 12.6 mmol) in 2N H₂ SO₄ (50 mL) and DME (50mL) was heated for 24 hr under N₂. The reaction mixture was concentratedto one half its volume in vacuo, diluted with H₂ O (100 mL) andextracted with CH₂ Cl₂ (5×50 mL). The organic layer was dried (Na₂ SO₄)and evaporated to yield a solid which was crystallized from CH₂ Cl₂-hexane to yield 2.68 g (80%) of 11 as a light brown solid: mp 185°-187°C.; ν_(max) (CHCl₃) 1750 (shoulder, ketone), 1745 (lactone), 1660 cm⁻¹(pyridone); ¹ H-NMR (CDCl₃) δ0.91 (t, 3, J=7 Hz, CH₂ CH₃), 1.80 (q, 2,J=7 Hz, CH₂ CH₃), 2.93 (t, 2, J=6.5 Hz, CH₂ C=0), 4.30 (t, 2, J=6.5 Hz,CH₂ N), 5.35 (m, 2, ArCH₂ O), 7.17 (s, 1, aromatic H). Anal. Calcd forC₁₃ H₁₃ NO₅ : C, 59.32; H, 4.94; N, 5.32. Found: C, 59.12, H, 4.91; N,5.16.

Synthesis of Camptothecin Analogs Synthesis of11-hydroxy-20(RS)-camptothecin

11-hydroxy-20(RS)-camptothecin is prepared from11-methoxy-20(RS)-camptothecin by demethylation of the latter withhydrobromic acid as follows:

11-Methoxy-20(RS)-camptothecin

A mixture of 4-methoxy-2-aminobenzaldehyde (180 mg, 1.19 mmol) and thetricyclic ketone 11 (300 mg, 1.14 mmol) in toluene (18 mL) was heatedunder N₂ in a flask equipped with a Dean-Stark trap. At refluxp-toluenesulfonic acid (5 mg) was added, and the red-brown solution washeated for an additional 2 hr. The toluene was removed under reducedpressure to give a brown solid which was treated with water (10 mL) andchloroform (20 mL). The aqueous phase was extracted with additionalchloroform (3×20 mL) and the combined extracts dried (Na₂ SO₄).Evaporation gave a brown solid which was recrystallized frommethanol-chloroform to give 216 mg (50%) of compound as a tan solid:275°-279° C.; mass spectrum (electron impact), m/z 378.1219M⁺ ; C₂₁ H₁₈N₂ O₅ requires 378.1214; ν_(max) (KBr) 3480 (OH), 1745 (lactone), 1660(pyridone), 1622, 1236 and 1152 cm⁻¹ ; ¹ H-NMR (DMSO-d₆) δ0.87 (t, 3,J=7 Hz, H-18), 1.85 (m, 2, H-19), 3.95 (s, 3, 11-OCH₃), 5.24 (s, 2,H-5), 5.42 (s, 2, H-17), 7.32 (s, 1, H-14), 7.37 (dd, 1, J=9, 2.5 Hz,H-10), 7.56 (d, 1, J=2.5 Hz, H-12), 8.02 (d, 1, J=9 Hz, H-9), 8.60 (s,1, H-7).

11-Hydroxy-20(RS)-camptothecin

11-methoxy-20(RS)-camptothecin (75 mg) was combined with 48% aqueous HBr(2.5 mL) and heated at reflux for 6 hr. The red-brown mixture wasstripped of solvent under high vacuum. Chromatography of the residuethrough silica gel (15 g) (7% MeOH-CHCl₃) gave the 11-hydroxy compound(33 mg, 45%) which was further purified by recrystallization from 13%MeOH in CHCl₃ : mp 323°-326° C.; mass spectrum (electron impact), m/z364.1054 M⁺, C₂₀ H₁₆ N₂ O₅ requires 364.1059; ν_(max) (KBr) 3450, 1742,1654, 1613, 1592, 1570, 1245 cm⁻¹ ; ν_(max) (EtOH), 224 (log ε 4.58),259, (4.39), 353 (4.16), 371 (4.19), 387 (4.20); ¹ H-NMR (DMSO-d₅):δ0.88 (t, 3, J=7 Hz, H-18), 1.85 (m, 2, H-19), 5.20 (s, 2, H-5), 5.41(s, 2, H-17), 6.51 (br s, 1, OH-20 ), 7.26 (dd, 1, J=9, 2.5 Hz, H-10),7.28 (s, 1, H-14).

10-Hydroxy-20(RS)-camptothecin

This compound is prepared in a manner analogous to that described forthe 11-hydroxycamptothecin using 5-methoxy-2-aminobenzaldehyde which isreacted with the tricyclic ketone 11 in the presence ofp-toluenesulfonic acid. The product is 10-methoxy-20(RS)-camptothecinwhich on treatment with refluxing hydrobromic acid as described for11-hydroxy-camptothecin, gives 10-hydroxy-20(RS)-camptothecin.

9-Methoxy-20(RS)-camptothecin and 9-Hydroxy-20(RS)-camptothecin

In a manner analogous to that described for11-methoxy-20(RS)-camptothecin, 6-methoxy-2-aminobenzaldehyde is treatedwith the tricyclic 11 ketone in the presence of p-toluenesulfonic acidyielding 9-methoxy-20(RS)-camptothecin. Demethylation with hydrobromicacid gives 9-hydroxy-20(RS)-camptothecin.

10-Nitro-20(RS)-camptothecin

A mixture of 2-amino-5-nitrobenzaldehyde (95 mg, 0.57 mmol) and thetricyclic ketone 11 (150 mg, 0.57 mmol) was heated at 120° C. for 10min. The temperature was raised to 160° C., and the dark molten mass waskept at this temperature for 1.5 hr with occasional stirring.Chromatography of the residue through silica gel (20 g) using 0.5% MeOHin CHCl₃ afforded the title compound (108 mg) as a yellow solid; mp297°-300° C. (decomp.); mass spectrum (electron impact), m/z 393.0965M⁺, C₂₀ H₁₅ N₃ O₆ requires 393.0960; ν_(max) (KBr) 3450 (OH), 1745(lactone), 1660 (pyridone), 1620, 1350, and 1160 cm⁻¹ ; ¹ H-NMR (TFA-d₁)δ1.14 (t, 3, J=7 Hz, H-18), 2.15 (m, 2, H-19), 5.88 (s, 2, H-5), 5.68(Abq, 2, J=17 Hz, Δγ=85 Hz, H-17), 8.43 (s, 1, H-14), 8.70 (d, 2, J=8Hz, H-12), 9.05 (d, 2, J=8 Hz, H-11), 9.35 (s, 1, H-9), 9.60 (s, 1,H-7).

10-Amino-20(RS)-camptothecin

A suspension of 10-nitro-20(RS)-camptothecin (100 mg) and 10% Pd/C (40mg) in absolute EtOH (40 mL) was stirred in an atmosphere of H₂ at roomtemperature for 30 min. Filtration through Celite and removal of thesolvent under reduced pressure gave a tan yellow solid (86 mg crude).Recrystallization from 13% MeOH/CHCl₃ gave the pure product (30 mg) asan olive-yellow solid: mp, softening at 135° C., gradual blackening uponfurther heating; mass spectrum (electron impact), m/z 363.116 M⁺ ; C₂₀H₁₇ N₃ O₄ requires 363.1218; ν_(max) (KBr) 3440 (OH, NH₂), 1750(lactone), 1660 (pyridone) cm⁻¹ ; ¹ H-NMR (TFA-d) δ1.06 (t, 3, J=7 Hz,H-18), 2.08 (d, J=7 Hz, H-17), 5.89 (s, 2, H-5), 5.70 (Abq, 2, J=17 Hz,Δγ=85 Hz, H-17), 8.34 (d, J=9 Hz, H-12), 8.64 (d, J= 9 Hz, H-11), 9.26(s, 1, H-(9), 9.43 (s, 1, H-7).

9-Nitro-20(RS)-camptothecin and 9-Amino-20(RS)-camptothecin

A mixture of 2-amino-6-nitrobenzaldehyde is treated with the tricyclicketone 11 in the manner described for the 10-nitro series above yielding9-nitro-20(RS)-camptothecin. This compound, after reduction withpalladium/carbon, yielded 9-amino-20(RS)-camptothecin. Alternatively,the 9-amino compound is obtained in one step by reaction of2,6-diaminobenzaldehyde with ketone 11.

11-Nitro-20(RS)-camptothecin and 11-Amino-20(RS)-camptothecin

In a manner similar to that described for 10-nitro-20(RS)-camptothecin,a mixture of 2-amino-4-nitrobenzaldehyde is treated with the tricyclicketone 11 yielding 11-nitro-20(RS)-camptothecin which in turn is reducedto 11-amino-20(RS)-camptothecin by palladium/carbon. Alternatively, the11-amino-20(RS)-camptothecin is obtained by reaction of2,4-diaminobenzaldehyde with ketone 11.

10,11-Dihydroxy-20(RS)-camptothecin

A solution of the crude dibenzyloxy aminoacetal (400 mg) and thetricyclic ketone 11 (132 mg, 0.5 mmol) in toluene (60 mL) was refluxedfor 8 hr. It was filtered hot, and the pure dibenzylether was collectedupon cooling (200 mg, 81%); mp 276° C. ν_(max) (KBr) 3440, 1740, 1650,1590, 1490, 1440, 1380, 1250, 1140, 1100 cm⁻¹ ; 250 MHz ¹ H-NMR(DMSO-d₆) δ0.88 (t, 3, J=7 Hz, H-18), 1.86 (m, 2, H-19), 5.22 (s, 2,H-17), 5.34 (s, 2, 10-OCH₂ --C₆ H₅), 5.39 (s, 2, 11-OCH₂ --C₆ H₅), 5.41(s, 2, H-5), 6.5 (s, 1, OH), 7.25 (s, 1, H-14) 7.35-7.65 (m, 12, H- 9,12, --OCH₂ --C₆ H₅), 8.44 (s, 1, H-7). Anal. calcd for C₃₄ H₂₈ N₂ O₆ :C, 72.84; H, 5.03; N, 5.00. Found C, 72.91; H, 5.09; N, 4.96.

The dibenzyl ether (130 mg, 0.23 mmol) was mildly refluxed for 2 hr in24% HBr (50 mL). The acid was removed, and the residue was dissolved inhot methanol (50 mL). Ether (50 mL) was added at room temperature andthe yellow powdery dihydroxy camptothecin hydrobromide was collected(122 mg, 77%) mp>300° C. ν_(max) (KBr) 3400 (b), 1740, 1655, 1585, 1545,1510, 1395, 1300, 1270, 1200, 1160 cm⁻¹ ; ¹ H NMR (DMSO, d₆): δ0.88 (t,3, J=7 Hz, H-18), 1.85 (m, 2, H-19), 5.20 (s, 2, H-17), 5.42 (s, 2,H-5), 7.31 (s, 2, H-9, H-14), 7.40 (s, 1, H-12), 8.45 (s, 1, H-7). Anal.calcd for C₂₀ H₁₇ BrN₂ O.sub. 6. 0.5 H₂ O: C, 51.08; H, 3.86; N, 5.95;Br, 16.99. Found C, 51.09; H, 4.04; N, 5.78; Br, 16.83.

Dihydroxy hydrobromide salt (110 mg, 0.23 mmol) was suspended in water(10 mL). Sodium hydroxide (0.1 N, 7.2 mL) was added and the mixture wasagitated. The resulting clear solution was acidified using 5N HCl; andafter an hour, the sample was centrifuged, the supernatant liquid wasdecanted and the process repeated with additional water (20 mL). Theresidue was dried (78 mg, 74%); mp>300° C. ν_(max) (KBr): 3490, 3000(b), 1740, 1645, 1590, 1460, 1385, 1265, 1190, 1150 cm⁻¹. ¹ H NMR (DMSO,d₆): δ0.38 (t, 3, J=7 Hz, H-18), 1.87 (q, 2, H-19), 5.20 (s, 2, H-17),5.42 (s, 2, H-5), 7.35 (s, 1, H-14), 7.44 (s, 1, H-9), 7.52 (s, 1,H-12), 8.51 (s, 1, H-7). Anal. calcd for C₂₀ H₁₆ N₂ O₆. 0.75 H₂ O: C,61.06; H, 4.44; N, 7.12. Found C, 61.12; H, 4.44; N, 7.09.

10-Chloro-20(RS)-camptothecin

This compound was prepared by treating 5-chloro-2-aminobenzaldehyde withthe tricyclic ketone 11.

A solution of the 5-chloro-2-aminobenzaldehyde (80 mg, 0.51 mmol) andthe tricyclic ketone 11 (100 mg, 0.38 mmol) in toluene (60 mL) wasrefluxed for 15 min. p-Toluenesulfonic acid (10 mg) was then added, andrefluxing was continued for an additional 5 hr. The solvent was removedin vacuo and the residue chromatographed (silica gel 60, 2% MeOH-CHCl₃).The product obtained was recrystallized from CHCl₃ -MeOH-EtOAc; mp 270°C., 60 mg (41%). ν_(max) (KBr), 3430, 1745, 1655, 1600, 1495, 1230, 1160cm⁻¹. 250 MHz ¹ H-NMR (TFA-d₁) δ1.15 (t, 3, J=7 Hz, H-18), 2.16 (m, 2,H-19), 5.73 (ABq, 2, J=17 Hz, Δγ=85 Hz, H-17), 5.84 (s, 2, H-5), 8.29(d, 1, J= 9 Hz, H-11), 8.35 (s, 1, H-14), 8.40 (s, 1, H-9), 8.45 (d, 1,J=9 Hz, H-12), 9.31 (s, 1, H-7). Anal. calcd for C₂₀ H₁₅ ClN₂ O₄ 0.5 H₂O: C, 61.47; H, 4.12; N, 7.17; Cl, 9.07. Found C, 61.41; H, 4.12; N,7.12; Cl, 9.11.

10-Methyl-20(RS)-camptothecin

5-Methyl-2-aminobenzaldehyde was treated with the tricyclic ketone 11 togive the title compound.

The tricyclic ketone 11 (130 mg, 0.5 mmol) and the5-methyl-2-aminobenzaldehyde (560 mg) in toluene (60 mL) were refluxedfor 0.5 hr. Acetic acid (1 mL) and p-toluenesulfonic acid (35 mg) wereadded, an refluxing was continued for an additional 5 hr. The solventwas removed in vacuo, and the residue was triturated with warm ether (30mL). The product was recrystallized from chloroform-methanol-ether toyield pure compound (102 mg, 57%), mp 278°-280° C. (KBr) 3460, 2980,1740, 1655, 1590, 1550, 1470, 1450, 1370, 1260, 1240, 1160, 1050 cm⁻¹.250 MHz ¹ H-NMR (DMSO-d₆) δ0.89 (t, 3, J=7 Hz, H-18), 1.87 (q, 2, H-19),2.54 (s, 3, 10-CH₃), 5.24 (s, 2, H-17), 5.42 (s, 1, H-5), 7.31 (s, 1 ,H-14), 7.69 (d, 1, J=8.6 Hz, H-11), 7.86 (s, 1, H-9), 8.05 (d, 1, J=8.6Hz; H-12), 8.55 (s, 1, H-7). Anal. calcd for C₂₁ H₁₈ N₂ O₄. 0.25 H₂ O:C, 68.75; H, 5.08; N, 7.64. Found C, 68.74; H, 5.08; N, 7.64.

11-Formyl-20(RS)-camptothecin

2-Nitroterephthaldicarboxaldehyde was converted to the ethylene diacetalby conventional methods and reduced using Na₂ S. A solution of the nitrodiacetal (4.1 g, 17.5 mmol), Na₂ S (14 g) in 80% ethanol (15 mL) wasrefluxed for 1 hr. Ethanol was removed in vacuo, the reaction mixturewas diluted with water (10 Ml) and the aqueous phase was extracted withCH₂ Cl₂ (4·50 mL). The organic phase was washed with water, dried(MgSO₄), and evaporated to give the aminodiacetal, which wasrecrystallized from ethyl acetatehexane (2.8 g, 78%); mp 76° C. ν_(max)(KBr) 3480, 3395, 3000, 2960, 2900, 1625, 1445, 1395, 1085, 950 cm⁻¹. 60MHz ¹ H NMR (CDCl₃ -D₂ O) δ4.0 (m, 8, --OCH₂ CH₂ O--), 5.6 (s, 1,--O--CH--O--, C-4), 5.7 (s, 1, --O--CH--O--, C-1), 6.6 (s, 1, H-3), 6.65(d, 1, J=8 Hz, C-5), 7.2 (d, 1, J=8 Hz, H-6). Anal. calcd for C₁₂ H₁₅NO₄ : C, 60.66 ; H, 6.36; N, 5.90. Found C, 60.79; H, 6.41; N, 5.84.

A solution of the tricyclic ketone 11 (265 mg, 1.0 mmol), aminodiacetal(500 mg, 2.1 mmol), 300 mg initially, 100 mg each at intervals of 5 and10 hr) in toluene (70 mL) was refluxed for 0.5 hr. Acetic acid (2 mL)was added and refluxing continued for 18 hr. The solvent was evaporatedin vacuo, and the residue was taken up in 75% methanol (250 mL). Conc.HCl (3 mL) was added and the reaction mixture heated at 50°-60° C. for24 hr. The mixture was filtered, and the residue was washed with waterand recrystallized from CHCl₃ -MeOH-EtOAc. mp: 276°-279° C. (175 mg,45%). ν_(max) (KBr) 3460, 1745, 1690, 1655, 1600, 1200, 1150, 1135 cm⁻¹.250 MHz ¹ H NMR (TFA-d₁), δL, 16 (t, 3, J=7 Hz, H- 18) 2.16 (q, 2, J=7Hz, H-19), 5.78 (ABq, 2, J=18 Hz, Δγ=85 Hz, H-17), 5.89 (s, 2, H-5),8.43 (s, 1, H-14), 8.66 (d, 1, J=8.5 Hz, H-10), 8.60 (d, 1, J=8.5 Hz,H-9), 9.12 (s, 1, H-12), 9.49 (s, 1, H-7), 10.42 (s, 1, CHO). Anal.calcd. for C₂₁ H₁₆ N₂ O₅. H₂ O: C, 64.01; H, 4.56; N, 7.11. Found C,64.08, H, 4.21; N, 6.84.

11-Cyano-20(RS)-camptothecin

A mixture of 11-formyl-20(RS)-camptothecin (225 mg, 0.6 mmol),hydroxylamine hydrochloride (50 mg, 0.72 mmol), sodium formate (90 mg,1.3 mmol), and formic acid (6 mL) was refluxed for 1.5 hr. The mixturewas evaporated to dryness in vacuo, and the residue was washed withwater, dried and chromatographed (silica gel 60, 0.5% MeOH-CHCl₃) andrecrystallized from CHCl₃ -EtOAc to yield the 11-cyano compound (65 mg,29%): mp 288° C. ν_(max) (KBr) 3400, 2235, 1735, 1655, 1590, 1450, 1400,1230, 1150, 1110, 1045 cm⁻¹. 250 MHz ¹ H NMR (DMSO-d₆): δ0.88 (t, 3, J=7Hz, H-18), 1.88 (m, 2, H-19), 5.32 (s, 2, H-17), 5.44 (s, 2, H-5), 7.37(s, 1, H-14), 7.98 (d, 1, J= 8.5 Hz, H-10), 8.32 (d, 1, J=8.5 Hz, H-9),8.74 (s, 1, H-12), 8.80 (s, 1, H-7). Anal. calcd for C₂₁ H₁₅ N₃ O₄. 1.5H₂ O: C, 62.99; H, 4.52; N, 10.49. Found C, 62.99; H, 3.95; N, 10.20.

Alternatively, 11-cyano-20(RS)-camptothecin can be prepared by thereaction of 5-cyano-2-aminobenzaldehyde with the tricyclic ketone 11.

Preparation Of Camptothecin Analogs With Modified A Ring Structure

The reaction of the tricylic ketone 11 with suitable precursors otherthan substituted ortho-amino-benzaldehydes can be used to give activenew camptothecin analogs exemplified by the following non-limitingexamples:

10-Aza-20(RS)-camptothecin

A solution of 4-aminonicotinaldehyde (24.2 mg, 0.198 mmol), thetricyclic ketone 11 (53.5 mg, 0.203 mmol) and p-TsOH. H₂ O (2 mg) intoluene (25 mL) was refluxed for 4 days using Dean-Stark trap. Thesolvent was removed under reduced pressure, and the residue waschromatographed through silica gel (20 g) using CHCl₃ -acetone-MeOH(5:1:1). The product was crystallized from 13% MeOH in CHCl₃ and EtOAc:mp 289°-292° C.; mass spectrum (electron impact), m/z 349.1061 M⁺ ; C₁₉H₁₅ N₃ O₄ requires 349.1066; ν_(max) (KBr) 3320 (OH), 1730 (lactone),1650 (pyridone), 1600 (aromatic) cm⁻¹ ; ¹ H NMR (CDCl₃) 1.05 (t, 3,J=7.8 Hz, H-18), 1.92 (m, 2, H-19), 5.35 (s, 2, H-5), 5.52 (ABq, 2, J=18Hz, Δγ=85 Hz, H-17), 7.74 (s, 1, H-14), 8.04 (d, 1, J=5.5 Hz, H-12),8.53 (s, 1, H-7), 8.84 (d, J=5.5 Hz, H-11), 9.4 (s, 1, H-9).

A-Nor-9-thia-20(RS)-camptothecin

This sulfur containing camptothecin analog is prepared by the reactionof 3-amino-2-formylthiophene with tricyclic ketone 11.

A solution of 3-amino-2-formylthiophene (79 mg, 0.62 mmol) and thetricyclic ketone 11 (96 mg, 0.37 mmol) in toluene (1.5 mL) was broughtto reflux and then cooled before adding a crystal of p-toluenesulfonicacid. The mixture was refluxed for 2.5 hr under N₂, cooled and theprecipitate filtered. The crude material was chromatographed on silicagel (20 g) by elution with 2% MeOH in CHCl₃. Crystallization of theproduct from 13% MeOH-CHCl₃ and EtOAc yielded the title compound as ayellow solid (19 mg, 15%): mp 297°-298° C.; ν_(max) 1740 (lactone), 1655cm⁻¹ (pyridone); ¹ H NMR (TFA-d₁) δ1.05 (t, 3, J=7 Hz, H-18), 2.07 (q,2, J=7 Hz, H-19), 5.60 (m, 2, H-17), 5.65 (s, 2, H- 5), 7.89 (d, J=6 Hz,H-11), 8.05 (s, 1, H-14), 8.57 (d, J=6 Hz, H-10), 9.23 (s, 1, H-7).Anal. (C₁₈ H₁₄ N₂ O₄ S), calcd. C, 61.02; H, 3.95; N, 7.91. Found C,60.65; H, 4.01; N, 7.78.

10,11-Methylenedioxy-20(RS)-camptothecin

The required ortho-aminoaldehyde was prepared by reduction of2-nitropiperonal. This compound (60 mg, 0.36 mmol) and the tricyclicketone 11 (53 mg, 0.20 mmol) were refluxed for 8 hr in toluene (30 mL)containing p-TsOH. H₂ O (8 mg). The solvent was removed in vacuo, thered residue absorbed onto Celite (1 g) and chromatographed throughsilica gel (10 g) using 3% MeOH in CHCl₃. Concentration of theappropriate fractions gave 10.11-methylenedioxy-20(RS)-camptothecin (36mg, 45%) as a pale tan solid. Crystallization of this material fromCHCl₃ gave the analytical sample as a cream-colored solid: mp>250° C.(decomp); ν_(max) (KBr) 1750 (lactone), 1655 (pyridone), 1585 cm⁻¹(aromatic); ¹ H NMR (TFA-d₁) δ1.15 (t, 3, J=7 Hz, H-18), 2.16 (q, 2, J=7Hz, H-19), 5.76 (ABq, 2, J=17 Hz, Δγ=85 Hz, H-17), 5.73 (s, 2, H-5),6.44 (s, 2, OCH₂ O), 7.55 (s, 1, H-14 ), 7.69 (s, 1, H-9), 8.16 (s, 1,H-12), 9.05 (s, 1, H-7). Anal. calcd for C₂₁ H₁₆ N₂ O₆ : 392.1008. Found392.1009 (C₂₁ H₁₆ N₂ O₆. 1.0 H₂ O).

Preparation Of Water Soluble Camptothecin Analogs

The following non-limiting examples of water soluble camptothecinanalogs were prepared as follows. The 9-glycinamido, 9-succinamido,9-(N-methylpiperazinocarbonylamino), 9-(N,N-diethylaminoethoxycarbonylamino), and 9-diethylaminoethoxy camptothecin analogs have thestructures shown below. ##STR10##

Synthesis of 9-Glycinamido-20(RS)-camptothecin Hydrochloride

9-Amino-20(RA)-camptothecin was prepared from2-amino-6-nitrobenzaldehyde and tricyclic ketone 11 by the processdescribed above. 9-(tert-Butoxycarbonylglycinamido)-20(RS)-camptothecin.

A stirred solution of 9-amino-20(RS)-camptothecin (88 mg, 0.242 mmol)and N-(tert-butoxycarbonyl) glycine (110 mg, 0.629 mmol) in dryN,N-dimethylformamide (10 ml) under nitrogen was treated withdicyclohexylcarboniimide (125 mg, 0.607 mmol) at room temperature. Afterstirring for 18 hr., the turbid white mixture was filtered to remove thedicyclohexylurea byproduct. The solvent was removed by high vacuumdistillation, and the tan-yellow residue was chromatographed throughsilica gel (25 g) using a stepwise gradient of 250 ml each ofchloroform, 1% methanol/chloroform, and 2% methanol/chloroform. Theappropriate fractions afforded 55 mg (44%) of the 9-tert-butoxycarboxylglycinamido compound as a yellow solid. Recrystallization from methanolprovided the sample as a beige solid, mp 208°-210° C. ν_(max) (KBr) 3360(br, OH, amide NH), 1750 (lactone), 1710 (carbamate), 1692 (amide), 1660(lactone), 1622, 1598, 1493, 1370, 1256, 1235, 1165, 1110, 1058, 1032,825 and 728 cm.sup. -1 ; ¹ H NMR (DMSO-d₆) δ0.89 (t, 3, J=7 Hz, H-18),1.44 (s, 9, C(CH₃)₃), 1.88 (m, 2, H-19), 3.92 (d, 2, J=6 Hz, COCH₂ N),5.29 (s, 2, H-5), 5.44 (s, 2, H-17), 6.53 (s, 1, OH), 7.19 (t, 1, J=6Hz, CH₂ NHCO), 7.37 (s, 1, H-14), 7.79 (d, 1, J=7 Hz, H-10), 7.85 (t, 1,J=7 Hz, H-11), 8.03 (d, 1, J=7 Hz, H-12), 8.79 (s, 1, H-7), and 10.20(s, 1, amide H). Anal. calcd. for C₂₇ H₂₈ N₄ O₇.H₂ O: C, 60.21; H, 5.61;N, 10.40. Found: C, 60.35; H, 5.64; N, 10.23.

9-Glycinamido-20(RS)-camptothecin Hydrochloride

The tert-butoxycarboxylglycinamide derivative from above (21 mg, 0.040mmol) was suspended in methylene chloride (10 ml) under nitrogen andthen dissolved by the addition of methanol (0.75 ml). The stirredsolution was chilled to 0° C. and treated over 5 min with a saturatedsolution of hydrogen chloride in anhydrous dioxane (4.5 ml) resulting ina turbid yellow solution. The stirred mixture was left to warm to roomtemperature, and after 2 hr. the solvents were removed under reducedpressured to give the deprotected compound as an orange-yellow solid (18mg). The sample was taken up in deionized water (5 ml) and the hazyyellow solution filtered through a 0.45 μm membrane filter to removeextraneous water-insoluble material. The clear filtrate was lyophilizedto provide the pure salt as a fluffy yellow solid (14 mg, 77%), mpdarkening above 245° C. with no melting up to 310° C. ν_(max) (KBr)2400-3650 cm⁻¹ (OH, amide H, amine HCl salt), 1742 (lactone), 1700(amide, 1658 (pyridone), 1590, 1550, 1495, 1234, 1163, 1110, 1050, 902820 and 720 cm⁻¹ ; ¹ H NMR (DMSO-d₆) δ0.89 (t, J=7.5 Hz, H-18), 1.89 (m,2, H-19), 4.03 (d, 2, J=5.4 Hz, COCH₂ N), 5.30 (s, 2, H-5), 5.44 (s, 2,H-17), 7.37 (s, 1, H-14), 7.86 (d, 1, J=7 Hz, H-12), 7.92 (t, 1, J=7 Hz,H-11), 8.07 (d, 1, J=7 Hz, H-10), 8.35 (br S, 3, NH₃ ⁺), 8.95 (s, 1,H-7, 10.88 (s, 1, amide H). Anal. calcd. for C₂₂ H₂₁ C1N₄ O₅.3H₂ O: C,51.71; H, 5.32 ; Cl, 6.94; N, 10.96. Found: C, 51.82; H, 5.23; Cl, 6.75;N, 10.61.

Synthesis of 9-Succinamido-20(RS)-camptothecin, Sodium Salt

The 9-succinamido derivative is synthesized from9-amino-20(RS)-camptothecin (synthesis described above) by the followingmethod.

9-Succinamido-20(RS)-camptothecin

A stirred suspension of 9-amino-20(RS)-camptothecin (400 mg, 1,102 mmol)and succinic anhydride (125 mg, 1.25 mmol) in pyridine (5 ml) undernitrogen was heated at 95° C. for 2 hr. The solvent was removed from thebrown solution by high vacuum distillation to give the crude amide as abrown gum. Purification was effected by chromatography through silicagel employing a solvent gradient from 5% methanol/chloroform to 50%methanol/chloroform. Evaporation of the appropriate fractions gave 272mg of the 9-succinamido compound as an orange-tan solid (53%), andrecrystallization from methanol gave the material as a light tan solid,mg 265°-270° C. (decomp). ν_(max) (KBr) 2600-3650 (OH, NH, acid), 1738(lactone), 1650 (pyridone), 1520-1610 (broad), 1485, 1400, 1232, 1160,1110, 1050, 818, 720 cm⁻¹ ; ¹ H NMR (DMSO-d₆) δ0.89 (t, 3, J=7 Hz,H-18), 1.88 (m, 2, H-19), 2.45-2.50 (m, 4, --NCOCH₂ CH₂ CO₂ H), 5.24 (s,2, H-5), 5.42 (s, 2, H-17), 6.53 (br s, 1, OH), 7.33 (s, 1, H-14), 7.77(t, 1, J=7 Hz, H-11), 7.85 (d, 1, J=7 Hz, H-12), 7.91 (d, 1, J=7 Hz,H-10), 8.83 (s, 1, H-7), 10.73 (br s, 1, amide H or CO₂ H). Calcd. for(M⁺ --CO₂ --H₂ O): 401.1375. Found: 401:1368. Calcd. for C₂₄ H₂₁ N₃O₇.2.5H₂ O: C, 56.68; H, 4.73; N, 8.45. Found: C, 56.69; H, 4.65; N,8.26.

Alternatively, the 9-succinamido derivative can be prepared byhydrolysis of its ethyl ester which is prepared by the following generalmethod:

9-Amino-20(RS)-camptothecin in dry N,N-dimethylformamide containingpyridine is reacted at 0°-10° C. with a slight excess of ethylsuccinylchloride in N,N-dimethylformamide solution. After work-up andchromatography on silica gel, a 75% yield of the 9-(ethyl)glycinamidederivative is obtained.

9-Succinamido-20(RS)-camptothecin, Sodium Salt

The preceding succinamide free acid (151 mg, 0.326 mmol) was suspendedin methanol (5 ml) and the stirred mixture treated dropwise at roomtemperature over 5 min with 0.1N aqueous sodium hydroxide solution (3.26ml, 0.326 mmol, 1 eq.). During the addition the solution became hazyyellow-orange, and at the end of the addition the pH was near 7. Themethanol was evaporated under reduced pressure, and the resultingaqueous solution was diluted with deionized water (6 ml). Filtrationthrough a 0.5 μm membrane filter was followed by lyophilization toprovide the 9-succinamido-20(RS)-camptothecin salt as a fluffyorange-yellow solid (145 mg, 92%), mp >200° C. (decomp). ¹ H NMR(DMSO-d₆); δ0.89 (t, 3, J=7 Hz, H-18), 1.88 (m, 2, H-19), 2.41-2.60 (m,4, --NCOCH₂ CH₂ CO₂ Na), 5.30 (s, 2, H-5), 5.43 (s, 2, H-17), 6.55 (brs, 1, OH), 7.33 (s, 1, H-14), 7.76 (t, 1, J=8 Hz, H-11), 7.87 (d, 1, J=8Hz, H-12), 8.16 (d, 1, J=8 Hz, H-10), 12.51 (br s, 1, amide H).

Synthesis of 9-Diethylaminoethoxy-20(RS)-camptothecin Hydrochloride

The title compound is prepared from 9-hydroxy-20(RS)-camptothecin(synthesis of hydroxy-camptothecins described on pp. 30-32 of currentPatent Application) in the following manner:

9-Diethylaminoethoxy-20(RS)-camptothecin

A stirred mixture of 9-hydroxy-20(RS)-camptothecin (20 mg, 0.055 mmol),N,N-diethylaminoethylchloride hydrochloride (15.4 mg, 0.090 mmol), andpowdered anhydrous potassium carbonate (38 mg, 0.276 mmol) in anhydrousdimethylformamide (0.5 ml) was heated under nitrogen at 55° C. for 3 h.During the reaction, the mixture became clear yellow-orange and thentan. The solvent was removed by high vacuum distillation, and the tanresidue was dispersed on Celite and chromatographed through silica gel(5 g) using 10% methanol/chloroform. Evaporation of the appropriatefractions gave 14 mg (55%) of the desired aminoether as a pale yellowsolid. Recrystallization from ethyl acetate afforded the pure titlecompound as a beige solid, mp 173°-176° C. (decomp) γ_(max) (KBr)3150-3650 (br, OH), 2967 and 2920 (CH), 1745 (lactone), 1658 (pyridone),1592-1620 (aromatic), 1469, 1460, 1384, 1370, 1267, 1232, 1190, 1157,1110, 1050, 810 and 720 cm⁻¹ ; ¹ H NMR (DMSO-d₆) δ0.89 (t, 3, J=7 Hz,H-18), 1.11 (t, 6, J=7 Hz, --N(CH₂ CH₃)₂), 1.89 (m, 2, H-19), 2.82 (brs, 4, --N(CH₂ CH₃)₂ ; with D₂ O exchange, signal is at 2.88δ as aquartet, J=7 Hz), 3.17 (br s, 2, --OCH₂ CH₂ NEt₂ ; with D₂ O exchange,signal is at 3.24δ as a fine triplet), 4.37 (m, 2, --OCH₂ CH₂ NEt₂),5.29 (s, 2, H-5), 5.43 (s, 2, H-17), 6.53 (s, 1, 20-OH), 7.19 (d, 1, J=7Hz, H-10), 7.33 (s, 1, H-14), 7.76 (m, 2, H-11 and H-12), 8.87 (s, 1,H-7). Anal. calcd. for C₂₆ H₂₉ N₃ O₅ : 463.2107; found: 463.2119. Calcd.for C₂₆ H₂₉ N₃ O₅.2.0 H₂ O: C, 62.52; H, 6.65; N, 8.41; found: C, 62.40;H, 6.75, N, 8.62.

Alternatively, similar yields of the title compound can be realized byreaction in refluxing acetone containing a catalytic amount of sodiumiodide.

9-Diethylaminoethoxy-20(RS)-camptothecin Hydrochloride

The free base aminoether prepared as described above (275 mg) wassuspended in methanol (4 ml) at room temperature and treated dropwisewith 0.1N aqueous hydrochloric acid until pH 3 was achieved. The hazyorange solution was evaporated in a nitrogen stream, redissolved indeionized water (50 ml) and filtered through a 0.45 μm membrane. Theclear yellow solution was frozen and lyophilized to afford the titlecompound as a bright yellow fluffy solid (273 mg), mp 232°-235° C.γ_(max) (KBr) 2400-3650 (br irregular, OH, amine HCl salt, CH), 1745(lactone), 1658 (pyridone), 1592 and 1620 (aromatic), 1465, 1400, 1370,1266, 1232, 1193, 1014, 811 and 720 cm⁻¹ ; ¹ H NMR (DMSO-d₆) δ0.89 (t,3, J=7 Hz, H-18), 1.33 (t, 6, J=7 Hz, --N(CH₂ CH₃)₂), 1.88 (m, 2 ,H-19), 3.34 (m, 4, --N(CH₂ CH₃)₂, 3.63 fine t, 2--NCH₂ CH₂ O--), 4.63(fine t,2, Et₂ NCH₂ CH₂ O--), 5.28 (s, 2, H-5), 5.53 (s, 2, H-17), 7.22(m, 1, H-10), 7.33 (s, 1, H-14), 7.79 (m, 2, H-11 and H-12), 9.05 (s, 1,H-7), 10.46 (br s, 1, .tbd.N.sup.⊕ H). Anal. calcd. for C₂₆ H₃₀ ClN₃O₅.1.5 H₂ O: C, 59.26; H, 6.31; N, 7.97; Cl, 6.73. Found: C 59.48; H,6.27; N, 7.81; Cl, 7.06.

Synthesis of 9-(4-methylpiperazino) carbonylamino-20(RS)-camptothecinHydrochloride

The title compound was prepared from 9-amino-20(RS)-camptothecin(synthesis of the amino-camptothecins is described on pp. 33 and 34 ofcurrent patent application) in the following manner:

9-(4-Methylpiperazino)carbonylamino-20(RS)-camptothecin.

The 9-amino-20(RS)-camptothecin was added to chloroform (treated withalumina to remove hydroxylic components) containing triethylamine. Theresulting solution was treated with phosgene gas and filtered to removesolids. The filtrate containing the intermediate carbamoyl chloride wastreated with N-methylpiperazine under nitrogen and left overnight. Theturbid mixture was washed several times with aqueous sodium bicarbonatesolution, dried and evaporated to afford the crude title compound.Chromatography on silica gel provided9-(4-methylpiperazino)carbonylamino-20(RS)-camptothecin.

9-(4-Methylpiperazino) carbonylamino-20(RS)-camptothecin Hydrochloride

The preceding free base urea was suspended in methanol and treated withone equivalent of dilute aqueous hydrochloric acid. The methanol wasevaporated and the aqueous residue filtered through a membrane filter.The sample was lyophilized to provide the title compound.

Synthesis of 9-(N,N-Diethylaminoethoxy)carbonylamino-20(RS)-camptothecin Hydrochloride

The title compound was prepared from 9-amino-20(RS)-camptothecin in thefollowing manner:

9-(N,N-Diethylaminoethoxy) carbonylamino-20(RS)-camptothecin

The intermediate 9-carbamoyl chloride was prepared as in the precedingexample. The resulting chloroform solution was treated withN,N-diethylaminoethanol under nitrogen. After standing overnight, themixture was washed with aqueous sodium bicarbonate solution, dried andevaporated to afford the crude carbamate. Purification by silica gelchromatography gave the pure title carbamate as the free base.

9-(N,N-Diethylaminoethoxy)carbonylamino-20(RS)-camptothecinHydrochloride

The free base from the preceding example was suspended in methanol andtreated with one equivalent of dilute aqueous hydrochloric acid. Themethanol was evaporated and the aqueous solution filtered (membrane).Lyophilization afforded the water soluble title carbamate.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A pharmaceutical composition comprising acamptothecin having the structure shown below ##STR11## wherein R_(n) is(a) an amino acid amido group obtained by reaction of an amino group atthe 9, 10, 11 or 12-position of the A-ring with the carboxylic acidgroup of a naturally occurring α-amino acid, (b) a C₄₋₁₀ carboxylic acidamido group, (c) a urea group obtained by reaction of an amino group atthe 9, 10, 11 or 12-position of the A-ring with phosgene followed byreaction with a diamine, (d) a urethane group obtained by reaction of anamino group at the 9, 10, 11 or 12-position of the A-ring with phosgenefollowed by reaction with a tertiary-amino alcohol, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 2. The composition of claim 1, wherein said aminoacid amido group is obtained by reaction of an amino group at the 9, 10,11 or 12-position of the A-ring with the carboxylic acid group of anaturally occurring α-amino acid.
 3. The composition of claim 2, whereinsaid amino acid amido group is the glycinamido group.
 4. The compositionof claim 1, wherein said carboxylic acid amido group is the succinamidogroup.
 5. The composition of claim 1, wherein said urea group isobtained by reaction of an amino group at the 9, 10, 11 or 12-positionof the A-ring with phosgene followed by a reaction with a diamine. 6.The composition of claim 1, wherein said urea group is theN-methylpiperazinocarbonylamino group.
 7. The composition of claim 1,wherein said urethane group is obtained by reaction of an amino group atthe 9, 10, 11 or 12-position of the A-ring with phosgene followed by areaction with a tertiary-amino alcohol.
 8. The composition of claim 1,wherein said urethane group is the N,N-diethylaminoethoxycarbonylaminogroup.
 9. A method of inhibiting the activity of the enzymetopoisomerase I by contacting said enzyme with a camptothecin having thestructure shown below ##STR12## wherein R_(n) is (a) an amino acid amidogroup obtained by reaction of an amino group at the 9, 10, 11 or12-position of the A-ring with the carboxylic acid group of a naturallyoccurring α-amino acid, (b) a C₄₋₁₀ carboxylic acid amido group, (c) aurea group obtained by reaction of an amino group at the 9, 10, 11 or12-position of the A-ring with phosgene followed by reaction with adiamine, (d) a urethane group obtained by reaction of an amino group atthe 9, 10, 11 or 12-position of the A-ring with phosgene followed byreaction with a tertiaryamino alcohol, or a pharmaceutically acceptablesalt thereof.
 10. The method of claim 9, wherein R_(n) is an amino acidamido group.
 11. The method of claim 9, wherein R_(n) is a C₄₋₁₀carboxylic acid amido group.
 12. The method of claim 9, wherein R_(n) isa urea group.
 13. The method of claim 9, wherein R_(n) is a urethanegroup.